Yeast strain having improved capability for fermenting xylose in the presence of acetic acid

ABSTRACT

The invention relates to a method for selecting a yeast strain having improved capability for fermenting a pentose, advantageously xylose, in the presence of organic acid, advantageously acetic acid, in non-dissociated form, in which at least one yeast strain that is capable of fermenting said pentose is consecutively cultured in the following two media: a first growth medium comprising said pentose as the only carbon source and said organic acid in non-dissociated form; a second growth medium comprising another carbon source as the only carbon source, advantageously glucose, free of said organic acid in non-dissociated form, the consecutive culture in at least said two growth media being repeated at least twice, in the presence of rising concentrations of organic acid in non-dissociated form.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/568,188, filed Oct. 20, 2017, which in turn is a U.S. national phasepursuant to 35 U.S.C. § 371 of International Patent Application No.PCT/FR2016/050987, filed Apr. 27, 2016, which claims priority to FrenchPatent Application No. 1553760, filed Apr. 27, 2015, the disclosures ofwhich are all herein incorporated by reference in their entireties.

DOMAIN OF THE INVENTION

The present invention deals with yeast strains able to ferment pentoses,in particular xylose, including in the presence of inhibitors of thisfermentation including acetic acid in non-dissociated form.

More specifically, the present invention proposes a method with which toselect improved strains that are very effective in their capability tometabolize this type of sugar present in lignocellulose hydrolysates.

DESCRIPTION OF THE PRIOR ART

Lignocellulose or plant biomass, mainly coming from agricultural andagro-industrial activity, is a complex substrate, made of three mainfractions which are cellulose, hemicellulose and lignin. It is a matterof potentially recyclable waste, useful for production of ethanol, whosedemand is continuing to increase, in light for example of its use as abiofuel.

The method for producing ethanol from lignocellulose biomass consists ofrecovering as much of the sugars present in the cellulose andhemicellulose fractions as possible by hydrolysis and then transformingthem into ethanol by fermentation.

As for the fermentation of the sugars present in this biomass, includingboth C6 sugars (hexoses) and C5 sugars (pentoses), anaerobicfermentation by yeasts is preferred, in particular using Saccharomycescerevisiae, whose capability for fermenting glucose is well controlledand developed.

However, full attention is given to the fermentation of pentoses, inparticular xylose, which can represent up to 25 to 40% of the totalsugars contained in the lignocellulose biomass. Thus, yeast strains ableto ferment glucose were modified to also be able to metabolize pentoses.

As an example, document WO 2010/000464 reports obtaining yeast strainsable to ferment pentoses because of a bacterial gene coding for a xyloseisomerase (XI) which converts xylose into xylulose which can bemetabolized by the yeast.

As an alternative, a eukaryotic pathway should be noted comprisingxylose reductase (XR or XYL1) generating xylitol and a xylitoldehydrogenase (XDH or XYL2) that can also produce xylulose.

Thus, document WO 2012/072793 describes improved yeast strains combiningexogenous genes coding a xylose isomerase and a xylitol dehydrogenasewith which to eliminate xylitol which proves to be an inhibitor ofxylose isomerase. Such strains, in particular the strain deposited atthe CNCM (Collection Nationale de Cultures de Microorganismes) on Oct.5, 2011 under number I-4538, have improved yields and therefore provenindustrial utility for the production of ethanol.

Another crucial problem was showing, in the lignocellulose hydrolysates,fermentation inhibitors, among them are furaldehydes (furfural and HMF),phenolic compounds and organic acids (acetic acid, levulinic acid andformic acid). The presence of high concentrations of acetic acid, over 5g/kg (initial medium) and which can reach 10 g/kg, is intrinsicallylinked to the concentration of acetyl groups covalently bonded tohemicellulose molecules.

Prior work has taken on the improvement of the resistance of the strainsto the presence of acetic acid in the fermentation musts. Thus, documentWO 2011/080411 reported obtaining yeast strains whose resistance toacetic acid on glucose was improved.

However, acetic acid is also an inhibitor of xylose fermentation. Thisinhibition is characterized by a reduction of the xylose consumptionkinetics (Bellisimi et al., FEMS Yeast Res., 2009.9:358-364), whereaswith glucose, this inhibition is seen as a delay during initiation offermentation with the kinetics subsequently remaining unchanged. Itshould be noted that in the presence of both glucose and xylose in themedium, yeast strains ferment glucose first because of cataboliterepression.

Thus documents WO 2013/178915 and WO 2013/178918 describe methods forobtaining yeast strains able to metabolize pentoses, in particularxylose, and resistant to fermentation inhibitors, in particular aceticacid.

There is however a clear need to obtain new yeast strains able toferment hexoses and pentoses with a further reduced impact offermentation inhibitors, in particular acetic acid, notably on thefermentation kinetics of pentoses such as xylose.

DESCRIPTION OF THE INVENTION

The present invention rests on the inventor showing the possibility ofisolating new yeast strains having resistance against fermentationinhibitors, in particular acetic acid, not only involving their glucosemetabolism but also involving fermentation of pentoses, includingxylose.

In that way and in the context of the invention, a method was developedfor selecting yeast strains showing improved fermentation of pentoses,in particular xylose, in the presence of organic acid-type fermentationinhibitors, in particular acetic acid. Using such a method, it waspossible to isolate strains having improved xylose fermentationkinetics, in terms of xylose consumption rate, including in the presenceof high acetic acid concentrations.

Prior work investigating this problem should be noted:

Thus the document (Wright et al., FEMS Yeast Res., 2011. 11: 299-306)reports the improvement of the resistance of strains against acetate onxylose. This work tends to show that the phenomenon of resistanceagainst acetic acid on xylose could be an inducible process. What ismore, the results presented appear to be extremely mixed:

The culture selected by the SBR method (“Single Batch Repeat”) or with axylose-limited chemostat only show a small reduction in the final xyloseconcentration and consequently a small increase in ethanol concentrationduring the first three days, compared to the results from the startingstrain.

Further, the peak of the specific xylose consumption rate as a functionof the acetic acid concentration peaks at a value very close to thatobserved for the starting strain, 2.5 g/L.

Further, it is observed that with the isolated strain, the specificxylose consumption rate next decreases in a way comparable to what wasobserved with the starting strain. According to the conclusions drawn,these results would indicate that a prolonged selection in SPR culturecannot lead to a stable phenotype with acetic acid tolerance.

Another document (Sanda et al., Bioresource Technology, 2011.102:7917-7924) reported implementation of directed evolution on alignocellulose hydrolysate containing glucose and xylose. However, theSBR techniques which serve to select the fastest multiplying strainslead to the selection of strains growing the fastest on glucose.

Despite failures from the prior art, the inventors developed a newselection method serving to arrive at yeast strains having the desiredproperties in terms of xylose fermentation in the presence of aceticacid.

According to a first aspect, the present invention relates to a methodfor selecting a yeast strain having improved capability for fermenting apentose, in the presence of organic acid, in non-dissociated form.

Thus, the method according to the invention serves to select, from anisolated strain or a mixture of strains, one strain having a selectiveadvantage in terms of growth on a medium containing said pentose andsaid organic acid in non-dissociated form.

The method, the subject of this application, can be implemented on anisolated strain, in particular on the following strains:

-   -   the strain deposited under the Budapest treaty with the CNCM on        May 16, 2013 under number I-4749;    -   the strain deposited under the Budapest treaty with the CNCM on        Dec. 12, 2013 under number I-4829;    -   the strain deposited under the Budapest treaty with the CNCM on        Apr. 9, 2015 under number I-4966.

In this scenario and without wanting to be held to any one theory, theselection pressure exerted by the consecutive cultures in the growthmedia defined below allows the strain to acquire phenotypic traitsnecessary for increasing its capability for fermenting a pentose in thepresence of organic acid in non-dissociated form.

According to another aspect, it is a mixture of yeast strains whichundergoes the method according to the invention. In this scenario, atleast one of the strains undergoes selection pressure as describedabove, so as to develop improved capability for fermenting a pentose inthe presence of an organic acid in non-dissociated form. Alternatively,the mixture of strains already contains a strain having an improvedcapability for fermenting a pentose in the presence of organic acid innon-dissociated form and the method according to the invention serves toenrich the mixture in this strain, because of the faster multiplicationthereof, and thus isolate or select said strain.

According to a specific embodiment, before their culture in thedifferent growth media according to the invention, the yeast strain ormixture of strains undergoes a prior step of mutagenesis which couldenhance the appearance of the desired phenotype. Conventionally, thismutagenesis can be achieved by exposure to a chemical agent or toradiation, in particular ultraviolet (UV) radiation. Advantageously“gentle” conditions are chosen, for example exposure of 100 to 500J/cm², for example 300 J/cm² of UV radiation at 254 nm.

According to the method from the invention, the yeast strain or mixtureof strains is cultivated consecutively in at least two growth media. Inthe context of this application, the expressions “growth medium” and“culture medium” are used indistinguishably for designating a mediumcomprising the ingredients necessary for the survival and evenmultiplication of the yeasts that are present.

The first growth medium is characterized in that it comprises, as theonly carbon source, the pentose for which improved fermentationcapability is sought. It advantageously involves xylose or arabinose,still more advantageously xylose. Advantageously, this first medium is aliquid medium.

Advantageously, the pentose concentration of the growth medium is thatcommonly implemented when it is used as the only carbon source,specifically included between 5 and 100 g/L in the case of a liquidmedium (5 and 100 g/kg in the case of a solid medium), advantageouslyincluded between 25 and 90 g/L, for example equal to 50 g/L. Accordingto a specific embodiment, the growth medium contains 50 g/L of xylose.

Said medium also includes the organic acid which could inhibitfermentation of said pentose. It advantageously involves acetic acid orformic acid, still more advantageously acetic acid.

It should be noted that it is known that only the non-dissociated ornon-ionized form of such acids have inhibition capability. In thecontext of the invention, “un-ionized or non-dissociated form” of acarboxylic acid is understood as the protonated form thereof. Inpractice, the form of such organic acids depends on the pH of the mediumin which they are incorporated. At a pH greater than the pKa of theacid, the acid will be mostly found in dissociated form or COO⁻ ions. Incontrast and at a lower pH, the majority form is the non-dissociated orunionized form (COOH). In the remainder of the invention, it will bespecified, in particular in connection with the quantities orconcentrations, whether only the non-dissociated form of the organicacid present is considered, or whether reference is made to the aceticacid added into the medium, encompassing dissociated and non-dissociatedforms depending on the pH of said medium.

Advantageously, the concentration of the organic acid, advantageously asacetic acid, in the non-dissociated form of the growth medium isincluded between 0.5 and 5 g/L in liquid medium (equivalent to 0.5 and 5g/kg in solid medium), advantageously between 1.3 and 2.6 g/L. Inpractice and as an example, this final range corresponds to aconcentration of acetic acid added to a growth medium at pH 5 includedbetween 3 and 6 g/L.

According to the invention, this first growth medium is implemented in agrowth cycle with at least two or even three media, said cycle beingrepeated at least twice. In a way adapted to the invention, during atleast two growth cycles, the concentration of organic acid innon-dissociated form is increased.

In other words, the consecutive culture in at least two growth mediadefined in connection with the invention is repeated twice, in thepresence of rising concentrations of organic acid, advantageously ofacetic acid, in non-dissociated form. Thus and in the presence of ncycles (with n greater than or equal to 2), at least two concentrationsare implemented, with the first concentration lower than the secondconcentration.

As an example and for 8 cycles of culture in the presence of aceticacid, a plan for increasing the concentration in the first growth mediumcan be the following:

-   -   1^(st) cycle: acetic acid concentration added to a medium at pH        5 equal to 3 g/L (or 1.3 g/L of acetic acid in dissociated        form);    -   2^(nd) cycle: acetic acid concentration added to a medium at pH        5 equal to 3 g/L (or 1.3 g/L of acetic acid in non-dissociated        form);    -   3^(rd) cycle: acetic acid concentration added to a medium at pH        5 equal to 3 g/L (or 1.3 g/L of acetic acid in non-dissociated        form);    -   4^(th) cycle: acetic acid concentration added to a medium at pH        5 equal to 4 g/L (or 1.7 g/L of acetic acid in non-dissociated        form);    -   5^(th) cycle: acetic acid concentration added to a medium at pH        5 equal to 4 g/L (or 1.7 g/L of acetic acid in non-dissociated        form);    -   6^(th) cycle: acetic acid concentration added to a medium at pH        5 equal to 4 g/L (or 1.7 g/L of acetic acid in non-dissociated        form);    -   7^(th) cycle: acetic acid concentration added to a medium at pH        5 equal to 5 g/L (or 2.15 g/L of acetic acid in non-dissociated        form);    -   8^(th) cycle: acetic acid concentration added to a medium at pH        5 equal to 6 g/L (or 2.6 g/L of acetic acid in non-dissociated        form).

Beyond these two ingredients, this growth medium is advantageously acomplete synthetic medium, suited to the growth of yeast and can containconventional ingredients such as salts, buffers, yeast extract or anyother source of nitrogen the yeast can metabolize, vitamins, etc. In thecontext of the invention, “synthetic medium” is understood to be amedium whose chemical composition is known.

According to a specific embodiment, other than pentose as the onlycarbon source and the organic acid, such a medium can include:

-   -   Yeast extract, advantageously at a concentration of 5 g/L;    -   Diammonium phosphate, advantageously at a concentration of 4.7        g/L;    -   Citric acid, advantageously at a concentration of 11.4 g/L;    -   Trisodium citrate, advantageously at a concentration of 13.5        g/L;    -   ZnSO₄, advantageously at a concentration of 21.2 mg/L;    -   MgSO₄ 7H₂O, advantageously at a concentration of 1 g/L;    -   Thiamine, advantageously at a concentration of 18.24 mg/L;    -   Pyridoxine, advantageously at a concentration of 5.28 mg/L;    -   Biotin, advantageously at a concentration of 1.76 g/L;    -   Pantothenate, advantageously at a concentration of 3.8 mg/L;    -   Niacin, advantageously at a concentration of 20 mg/L;    -   Meso-inositol, advantageously at a concentration of 50 mg/L;    -   Riboflavin, advantageously at a concentration of 1 mg/L;    -   Para-aminobenzoate, advantageously at a concentration of 1.2        mg/L;    -   Tween 80, advantageously at a concentration of 1 g/L.

Beyond the specific composition of this growth medium, the culture forthe yeast strain or mixture of strains is advantageously made understandard conditions favorable to the growth of yeasts, in particularSaccharomyces type, and their fermentation activity, specifically:

-   -   An acid pH advantageously included between 4 and 6, even 4.5 and        5.5, even more advantageously equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 32° C.;    -   Under gentle stirring, for example equal to 100 rpm;    -   Under reduced conditions of oxygen supply (under limited O₂). In        practice, the culture can be made in a flask stoppered using a        cap reducing the supply of O₂ in the medium while allowing        evacuation of the CO₂ produced.

Generally, the culture is stopped when the source of hydrolyzableglucidic carbon, in this case the pentose, has been completely consumed.In practice, and advantageously, the culture is left for at least 24hours, even several days, advantageously for seven days.

The second step of the method according to the invention consists oftransferring the yeasts cultivated in the first growth medium into asecond growth medium, which is advantageously liquid.Characteristically, this is distinguished from the first culture mediumby the presence of a different carbon source and by the absence oforganic acid, advantageously of acetic acid, in non-dissociated form.

In practice, this medium is favorable to the growth of yeasts and allowslifting adaptation phenomena to the benefit of the acquisition of stablemutations. Thus, this step of the method according to the invention isconsidered as a de-adaptation phase.

This growth medium is characterized in that it comprises, as only carbonsource, a carbon source different from that of the first culture medium,specifically a pentose, in particular a xylose. According to a specificembodiment, the carbon source from the second culture medium is a sourceof hydrolyzable glucidic carbon, advantageously a hexose, still moreadvantageously glucose. Advantageously, the concentration of this secondcarbon source, advantageously glucose, is included between 5 and 50 g/L,advantageously included between 5 and 20 g/L, for example equal to 20g/L.

Further and advantageously, it involves a rich synthetic growth medium,comprising for example:

-   -   yeast extract, advantageously at a concentration of 10 g/L;    -   peptone, advantageously at a concentration of 10 g/L.

Advantageously, it is therefore a matter of a rich medium allowing allstrains of yeast to multiply without any limitation which would belinked to any auxotrophy.

Beyond the specific composition of this second growth medium, theculture for the yeast strain or mixture of strains is advantageouslymade under standard conditions favorable to the growth of the yeast,specifically:

-   -   An acidic and stable pH, advantageously included between 4 and        6, for example equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 30° C.;    -   Under medium stirring, for example equal to 150 rpm;    -   In the presence of oxygen, specifically aerobiosis. In practice,        the culture can be made in a baffled flask stoppered by a porous        cap which allows the supply of O₂ in the medium while allowing        evacuation of the CO₂ produced.

There again, the culture is stopped when the source of hydrolyzableglucidic carbon, advantageously glucose, has been completely consumed.It should be noted that in practice the growth in the second medium isfaster than in the first medium because of the aerobiosis, the glucoseas carbon source and the absence of acetic acid. Thus andadvantageously, the culture is done over several hours, advantageously24 hours.

According to the invention, the culture of the yeasts consecutively inthe two growth media and under the conditions described aboveconstitutes one cycle.

According to a specific embodiment, a cycle in the method according tothe invention further comprises the passage of the yeast into a thirdgrowth medium, advantageously liquid, intended to select the cellscapable of respiration, specifically having functional mitochondria. Inpractice, this step, which can be implemented with each cycle or atleast once in the method, serves to overcome the appearance of “petites”whose respiratory-deficient phenotype can be disadvantageous in thecontext of industrial yeast production methods.

Advantageously, this third medium is a poor or minimum medium containingas the only carbon source a carbon which can only be used by cells whichretained functional mitochondria. In this case, one is speaking of asource of strict respiratory carbon, meaning a carbon sourcesystematically involving mitochondrial oxidation and not producingethanol. It can advantageously be glycerol or possibly ethanol. In otherwords such a medium is free of fermentable sugar. Advantageously, theglycerol concentration of the growth medium is that commonly used whenit is used as the only carbon source, specifically included between 5and 50 g/L, advantageously included between 10 and 50 g/L, for exampleequal to 10 g/L so as to obtain sufficient biomass for inoculating thefirst culture medium of the following cycle.

By definition, a minimum medium contains, other than a carbon source, anitrogen source, a potassium source, a phosphorous source, a sulfursource, a magnesium source, a calcium source, an iron source, a sourceof trace elements and of water.

A medium which can be used for preparation of this third culture mediummay include:

-   -   a base, such as DIFCO® yeast nitrogen base, advantageously at a        concentration of 3.4 g/L;    -   and optionally ammonium sulfate, advantageously at a        concentration of 5 g/L.

Beyond the specific composition of this third growth medium, the culturefor the yeast strain or mixture of strains is advantageously made understandard conditions favorable to the growth of yeasts, specifically:

-   -   An acid pH advantageously included between 4 and 6, even 4.5 and        5.5, for example equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 30° C.;    -   Under medium stirring, for example equal to 150 rpm;    -   In aerobiosis. In practice, the culture can be made in a baffled        flask stoppered by a porous cap which allows the supply of O₂ in        the medium.

There again, the culture is stopped when the carbon source,advantageously glycerol, has been completely consumed. It should benoted that in practice the growth in this medium is a priori less quickthan in the second medium because of the mineral source of nitrogen.Thus and advantageously, the culture is done over several hours,advantageously 48 hours.

As already mentioned, the method according to the invention ischaracterized in that the culture of the yeast strain, consecutively inthe two or three growth media described above, is iterated in the formof cycles.

The number of cycles is chosen so as to enhance the selection of thestrain having the desired characteristics, specifically an improvedcapability for fermenting a pentose in the presence of an organic acidinhibitor, while also limiting the number of cycles for avoiding theappearance of unsoundnesses in the yeast.

Thus and advantageously, the number of cycles is at least equal to twoand advantageously less than or equal to 10, for example equal to 8. Inother words, the consecutive culture in the at least two growth media asdefined is repeated at least two times, advantageously more than 2 timesbut less than 10 times, for example 8 times.

According to an advantageous embodiment of the method according to theinvention, the consecutive culture in at least the two growth media isrepeated at least twice, in the presence of rising concentrations oforganic acid, advantageously acetic acid, in non-dissociated form. Inother words, the method according to the invention is done in thepresence of at least two distinct concentrations with the firstconcentration less than the second. This concentration can be increasedgradually, possibly by thresholds. Thus and advantageously for theadaptation of the yeast strains, at least the first two cycles are doneat constant concentration.

According to another advantageous aspect, the concentration of organicacid, advantageously acetic acid, in non-dissociated form in the firstgrowth medium is included between 1.3 and 2.6 g/L.

When the desired number of cycles is reached and for obtainingdifferentiated clones, the culture is advantageously spread on a solidmedium, still more advantageously on a synthetic medium favorable to thegrowth of yeasts, such as the YPG medium. The seeding level is typically2000 cells per plate and the growth takes place at 30° C. for about 72hours.

In a specific embodiment, the performance of the clones isolated thisway is tested.

For this purpose, pre-cultures in liquid medium, advantageously in asynthetic medium suitable to the growth of yeast, such as the YPGmedium, can be prepared.

Advantageously, these pre-cultures allow the seeding of the selectionmedium, specifically a medium equivalent to the first growth mediumdescribed above, comprising as the only carbon source said pentose,advantageously xylose, and said organic acid, advantageously aceticacid, in non-dissociated form. Preferably, an average acetic acidconcentration is chosen, for example equal to 5 g/L for a medium with pH5.

Advantageously the selected clones are the ones having the best growthin this medium. The growth level can be determined by the analysis ofthe turbidity using an optical density measurement from aspectrophotometer at a wavelength included between 600 and 700 nm, forexample equal to 630 nm. In practice, the growth can be evaluated inDeepWell type microplates or a liquid medium under the conditions statedbelow, specifically:

-   -   An acid pH advantageously included between 4 and 6, even 4.5 and        5.5, even more advantageously equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 32° C.;    -   Under gentle stirring, for example equal to 100 rpm, or without        stirring;    -   Under reduced conditions of oxygen supply (under limited O₂ or        anaerobiosis);    -   A growth time of at least 24 hours, for example 72 hours.

The seeding rate must therefore be moderate to prevent a “titrating”effect of the yeast, for example equal to 0.25 g/kg equivalent DM(DM=dry material).

An important first criterion is therefore the capability of the one ormore selected yeast strains to grow in a medium containing a pentose, inparticular xylose, in the presence of an organic acid type inhibitor, inparticular acetic acid, in non-dissociated form.

According to another embodiment, the selection method can comprise astep for evaluation of the mass loss as a function of fermentation timein the selection medium and under conditions as defined above. Themeasurement of the mass loss from the fermentation flask is correlatedwith the production of alcohol. Tracking this mass loss thereforereflects the kinetics of pentose, advantageously xylose, fermentation inthe presence of an organic acid-type inhibitor, in particular aceticacid, and non-dissociated form.

Under these conditions and in the presence of only pentose as carbonsource (no glucose), the expected profile is single phase, with anassociated ax+b type kinetics when the sugar is in a nonlimitingconcentration (at the start of the culture). In the context of theselection of an advantageous strain, in particular for its pentose, inparticular xylose, fermentation rate, the value a (positive number) isas high as possible.

Further, a strain of yeast is sought capable of resisting acetic acid inxylose medium, and doing so including at high acetic acidconcentrations. Thus, the maximum CO₂ production rate can be measuredduring xylose fermentation on a medium as previously described butcontaining variable concentrations of acetic acid from the fermentationmedium.

For a given acetic acid concentration, it is preferred that the selectedstrain have a maximum xylose consumption speed, equal to twice the massloss by fermentation, greater than already isolated strains, and do sowhatever the acetic acid concentration (including in the absence ofacetic acid), advantageously in the concentration range of real media,for example between 4 and 8 g/kg.

Further and according to an advantageous embodiment, it is verified thatthe capability of the selected strain to ferment glucose (hexose) andxylose (pentose) in the presence of the organic acid, advantageouslyacetic acid, in non-dissociated form is not affected. It is thereforethen a matter of analyzing the fermentation characteristics in general,in a medium near real media containing both hexoses and pentoses.

In practice the fermentation is done on a YFP type rich medium,containing glucose, xylose and acetic acid, for example a mediumcomprising:

-   -   Yeast extract, advantageously at a concentration of 10 g/L;    -   Bacto-peptone, advantageously at a concentration of 10 g/L;    -   Glucose, advantageously at a concentration of 55 g/L;    -   Xylose, advantageously at a concentration of 45 g/L;    -   Acetic acid, advantageously with a quantity added to the medium        at a concentration of 5 g/L;    -   KOH.

The culture conditions are those suited to the growth and fermentationof yeast, advantageously:

-   -   An acid pH advantageously included between 4 and 6, even more        advantageously equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 32° C.;    -   Under gentle stirring, for example equal to 100 rpm;    -   Under reduced conditions of oxygen supply (under limited O₂ or        anaerobiosis);    -   A growth time of at least 24 hours, for example 72 hours.

The seeding rate must therefore be moderate to prevent a “titrating”effect of the yeast, for example equal to 0.25 g/kg equivalent DM(DM=dry material).

A profile is expected which shows the double effect of the acetic acidon the yeasts able to ferment xylose and glucose:

-   -   During the first phase, called the “glucose phase”, a delayed        initiation of the fermentation which is a short as possible,        advantageously under 24 hours, still more advantageously of        order 5 to 6 hours. Preferably, the selected strain has        therefore not lost resistance to acetic acid during glucose        fermentation.    -   During xylose fermentation, kinetics as favorable as possible,        meaning as fast and high as possible, with better performance        than the strains from the prior art.

According to an advantageous embodiment, the method according to theinvention is implemented on the strain of yeast having attractiveproperties in connection with the targeted application:

-   -   Good growth in synthetic and natural media;    -   Capability for fermenting glucose including, advantageously, in        the presence of an organic acid, in particular acetic acid, in        non-dissociated form;    -   Capability for fermenting xylose including, advantageously, in        the presence of an organic acid, in particular acetic acid, in        non-dissociated form;    -   And, advantageously, reduced inhibition of the glucose        fermentation pathway by acetic acid.

Such strains are for example chosen from the following group:

-   -   the strain deposited under the Budapest treaty with the CNCM on        Oct. 5, 2011 under number I-4538;    -   the strain deposited under the Budapest treaty with the CNCM on        May 16, 2013 under number I-4749;    -   the strain deposited under the Budapest treaty with the CNCM on        Dec. 12, 2013 under number I-4829;    -   the strain deposited under the Budapest treaty with the CNCM on        Apr. 9, 2015 under number I-4966.

Preferred strains are the strain deposited with the CNCM on Dec. 12,2013 under number I-4829 and/or the strain deposited with the CNCM onApr. 9, 2015 under number I-4966.

Preferably, the strains selected at the end of the method according tothe invention retain the characteristics of interest from the strainsused for implementing said method.

According to another aspect, the present invention relates to a yeaststrain obtained using the method described above.

Remarkably, such a strain has a capability for fermenting xylose in thepresence of acetic acid that is still unequaled.

This capability is advantageously measured under the followingconditions:

A pre-culture of the strain is made in liquid medium, advantageously asynthetic medium favorable to the growth of yeast, such as the YPGmedium, and under conditions described in connection with the secondmedium used in the method described above.

After 24 to 72 hours of culture at 30° C., the strain is transferredinto a growth medium such as the first growth medium of the methodaccording to the invention. Such a medium is advantageously defined asfollows:

-   -   Yeast extract, advantageously at a concentration of 5 g/L;    -   Diammonium phosphate, advantageously at a concentration of 4.7        g/L;    -   Citric acid, advantageously at a concentration of 11.4 g/L;    -   Trisodium citrate, advantageously at a concentration of 13.5        g/L;    -   ZnSO₄, advantageously at a concentration of 21.2 mg/L;    -   MgSO₄ 7H₂O, advantageously at a concentration of 1 g/L;    -   Thiamine, advantageously at a concentration of 18.24 mg/L;    -   Pyridoxine, advantageously at a concentration of 5.28 mg/L;    -   Biotin, advantageously at a concentration of 1.76 g/L;    -   Pantothenate, advantageously at a concentration of 3.8 mg/L;    -   Niacin, advantageously at a concentration of 20 mg/L;    -   Meso-inositol, advantageously at a concentration of 50 mg/L;    -   Riboflavin, advantageously at a concentration of 1 mg/L;    -   Para-aminobenzoate, advantageously at a concentration of 1.2        mg/L;    -   Tween 80, advantageously at a concentration of 1 g/L.

It additionally contains xylose as an only carbon source, at aconcentration advantageously equal to 50 g/L and variable concentrationsof acetic acid ranging preferably from 0 to 10 g/L so as to be able toestablish a dose-response curve. Here it involves values for quantitiesof acetic acid added to the culture medium at pH 5.

The conditions for seeding and culture are advantageously the following:

-   -   An acid pH advantageously included between 4 and 6, even 4.5 and        5.5, even more advantageously equal to 5;    -   A temperature included between 28 and 37° C., even between 30        and 35° C., advantageously equal to 32° C.;    -   Under gentle stirring, for example equal to 100 rpm;    -   Under reduced conditions of oxygen supply (under limited O₂ or        anaerobiosis);    -   A growth time of at least 24 hours, advantageously 72 hours;    -   A seeding rate advantageously equal to 0.25 g/kg equivalent DM        (DM=dry material).

At the end of this culture, the maximum CO₂ production rate isdetermined for each acetic acid concentration.

Under these conditions, the selection of yeast strains having thefollowing characteristics never obtained by strains from the prior artwas reported for the first time: The acetic acid dose needed to reducexylose consumption kinetics 50% is greater than 2.5 g/L in liquid medium(2.5 g/kg in solid medium), even 3 g/L, even greater than or equal to3.5 g/L, advantageously greater than 4 g/L, or even greater than orequal to 5 g/L. Here it involves values for quantities of acetic acidadded to the culture medium at pH 5.

In practice, faster xylose fermentation kinetics in real media,typically comprising from 2 and 8 g/kg of acetic acid, results. Thus andin the presence of a strain according to the invention, it is possibleto conceive of total fermentation of the sugars present in under 72hours, advantageously between 48 and 72 hours.

According to a particular embodiment, the yeast strain targeted by theinvention comprises at least one copy of an exogenous gene coding forxylose isomerase, advantageously from Clostridium phytofermentans. Inconnection with the invention, “exogenous” is understood to mean thefact that the gene comes from another organism.

According to another specific embodiment, the yeast strain targeted bythe invention comprises at least one supernumerary copy of the GAL2 genecoding for a hexose transporter also capable of capturing xylose.According to another embodiment, said strain comprises at least twosupernumerary copies of the GAL2 gene. This can be placed under thecontrol of a pADH1-type strong, constitutive promoter.

According to other specific embodiments, the yeast strain targeted bythe invention has one or more characteristics chosen from the followinggroup:

-   -   suppression of aldose reductase activity (GRE3);    -   overproduction of xylulokinase (XKS1), in particular by        modification of the promoter or introduction of supernumerary        copies;    -   expression or overproduction of the pentose phosphate pathway        (RPE1, RKL1, TKL1, TAL1, etc.);    -   absence of xylose reductase (XR) activity;    -   the capability for fermenting arabinose, advantageously because        of the insertion of a bacterial pathway such as described in WO        2008/041840 or European patent 1,499,708.

According to an advantageous embodiment, the yeast strain targeted bythe present invention belongs to the Hemiascomycetes group. Preferredstrains belong to the Saccharomyces, Candida, Pichia and Yarrowiagenera, advantageously Saccharomyces. Among Saccharomyces, itadvantageously involves Saccharomyces cerevisiae.

As shown in the context of the present application, a strain conformingto the invention is the strain deposited under the Budapest treaty atthe CNCM (Collection Nationale de Cultures de Microorganismes, InstitutPasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under numberI-4953 on Jan. 29, 2015.

In the context of the invention, “yeast strain” is understood to be agenetically rigorously identical population of yeast. This encompassesboth strains referred to as laboratory strains and those referred to asindustrial strains.

According to another aspect, the present invention also targets a yeastobtained by culture of a strain as defined above.

In the context of the invention, “yeast” is understood as a commercialproduct obtained by implementation of a production method for a yeaststrain. Thus, yeasts having different properties can be obtained from asingle strain, where these differences are connected with the productionmethod implemented.

According to another aspect, the invention relates to the use of astrain or yeast as defined above for the fermentation of a material,advantageously containing xylose and/or glucose, and/or for ethanolproduction. According to a specific embodiment, the material is alignocellulose material. Such material typically contains:

-   -   Pentoses, in particular D-xylose and L-arabinose;    -   Hexoses, in particular D-mannose, D-galactose, L-rhamnose and        D-glucose;    -   Uronic acids.

According to a specific aspect, the invention deals with a productionmethod for fermentation products or ethanol comprising the followingsteps:

-   -   Incubation of a material or medium containing xylose with a        strain or a yeast as defined above;    -   Fermentation under anaerobic or semi-anaerobic conditions;    -   Recovery of the one or more fermentation products, or ethanol.

According to a specific embodiment of this method, the material ormedium also contains glucose.

The present invention is going to be illustrated more ahead using thefollowing example embodiments, supported by the attached figures.However, they have no limiting scope.

LEGENDS FOR THE FIGURES

FIG. 1 shows the progression of the mass loss (expressed in g/kg) as afunction of fermentation time for various strains, in a mediumcontaining 50 g/L xylose and 5 g/L acetic acid at pH=5.

FIG. 2 shows the impact of acetic acid concentration (expressed in g/Lat pH 5) on the mass loss rate (production of CO₂ from xylose), duringfermentation of various strains, in a medium containing 50 g/L xylose.

FIG. 3 shows the progression of the mass loss (expressed in g/kg) as afunction of fermentation time for various strains, in a mediumcontaining 55 g/L glucose, 45 g/L xylose and 5 g/L acetic acid (at pH5).

EXAMPLE EMBODIMENTS

I) Material and Methods:

1) Strain:

Yeast: Saccharomyces cerevisiae

Strain: I-4829 or I-4966

2) Culture Medium and Conditions:

2-a) Directed Evolution:

Medium 1: YFX50=

-   -   Xylose 50 g/L;    -   Yeast extract 5 g/L;    -   Di-ammonium phosphate 4.7 g/L;    -   Citric acid 11.4 g/L;    -   Trisodium citrate 13.5 g/L;    -   ZnSO₄ 21.2 mg/L;    -   MgSO₄ 7H₂O 1 g/L;    -   Thiamine 18.24 mg/L;    -   Pyridoxine 5.28 mg/L;    -   Biotin 1.76 g/L;    -   Pantothenate 3.8 mg/L;    -   Niacin 20 mg/L;    -   Meso-inositol 50 mg/L;    -   Riboflavin 1 mg/L;    -   Para-aminobenzoate 1.2 mg/L;    -   Tween 80, 1 g/L.

The yeasts are cultivated in this medium with xylose as the only carbonsource and with acetic acid whose concentration increases over thecycles (see Results section).

The pH of the medium is kept at 5.

The culture is done at 32° C. with stirring at 100 rpm in flasksstoppered by caps which serve to reduce the supply of oxygen in themedium and allow CO₂ which is produced throughout this culture in excesspressure to escape. Under these conditions, the culture lasts aboutseven days.

Medium 2 (YPG):

-   -   10 g/L of yeast extract;    -   10 g/L de peptone;    -   20 g/L of glucose as the only carbon source.

The culture is done at 30° C. with stirring at 150 rpm in baffled flasksstoppered by porous caps which allow the supply of oxygen into themedium. Under these conditions, the culture lasts 24 hours.

Medium 3:

-   -   3.4 g/L of DIFCO® yeast nitrogen base;    -   5 g/L of ammonium sulfate;    -   10 g/L of glycerol as the only carbon source.

The culture is done at 30° C. with stirring at 150 rpm in baffled flasksstoppered by porous caps which allow the supply of oxygen into themedium. Under these conditions, the culture lasts 24 to 48 hours.

2-b) Selection of the Most Effective Strains:

At the end of the eighth cycle, the cells were spread at a concentrationof 2000 cells per dish (150 mm diameter) of agar medium (YPG+agar).

The resulting colonies were then cultivated in Deep Well format(=96−well microplate) in a YPG medium.

After 72 hours of culture at 30° C., the colonies were transferred intoa YFX50-Ac-5000 medium whose composition is indicated below:

YFX50-Ac-5000 g/kg mL/kg Distilled water qs 1000 Xylose 50 Yeast extracttype J 5 DAP 4.7 Citric acid 11.4 Trisodium citrate 13.5 Acetic acid 5pH 5 ZnSO₄ (10.6 g/L) 2 MgSO₄ 7H₂O (400 g/L) 2.5 Thiamine Vit B1 (18.24g/L) 1 Pyrodixine Vit B6 (5.28 g/L) 1 Biotin (1.76 g/L) + KOH 1Pantothenate (3.8 g/L) 1 Nicotinic acid (8 g/L) 2.5 Meso-inositol (50g/L) 1 Riboflavin (1 g/L) 1 Para-aminobenzoate (1.2 g/L) 1 Tween 80 1

In practice, it involves the same culture medium as the first growthmedium used in the method according to the invention, with an aceticacid concentration (quantity added into the culture medium at pH 5)equal to 5 g/L.

Likewise, the culture conditions are similar:

Conditions:

pH: 5

Temperature: 32° C.

O₂ conditions: without oxygen supply (anaerobic)

Stirring: 100 rpm

Length of the culture: 72 hours

Pre-Culture/Incubation:

-   -   Take up the colonies for the liquid cultures from Deep Well        (96-well micro plates);    -   0.25 g/kg equivalent DM for liquid cultures.        2-c) Validation of the Isolated Strains

Medium: YFP-5000

-   -   10 g/L of yeast extract;    -   10 g/L of bacto-peptone;    -   55 g/L of glucose;    -   45 g/L de xylose;    -   5 g/L of acetic acid (quantity added to the culture medium at pH        5);    -   KOH.        Conditions:

pH: 5

Temperature: 32° C.

O₂ conditions: without oxygen supply

Stirring: 100 rpm

Length of the culture: up to 72 hours

Pre-Culture/Incubation: 0.25 g/kg eq DM of yeast previously propagatedin YPG medium.

3) Evaluation of the Mass Loss:

The ethanol production is measured indirectly by a measurement of themass lost from the fermentation flask, since this mass loss is directlycorrelated with alcohol production. It is expressed in grams perkilogram of medium.

4) Evaluation of the CO₂Production Speed:

Starting with the determination of the mass loss (corresponding to CO₂production), it is possible to determine the xylose consumption rate(expressed in g/L) which is twice the mass loss.

The maximum CO₂ production rate is expressed in g·kg⁻¹·h⁻¹·g⁻¹ of DM(dry material).

II) Results:

1) Selection of Strains of Interest:

1-a) Directed Evolution:

The directed evolution was done in Single Batch Repeat mode:

-   -   Cycles 1 to 3: medium 1 with 3 g/L of acetic acid (added into        the medium at pH 5) and then medium 2 and then medium 3;    -   Cycles 4 to 6: medium 1 with 4 g/L of acetic acid and then        medium 2 and then medium 3;    -   Cycle 7: medium 1 with 5 g/L of acetic acid and then medium 2        and then medium 3;    -   Cycle 8: medium 1 with 6 g/L of acetic acid and then medium 2        and then medium 3;        1-b) Selection of the Most Effective Strains:

After isolation of the clones on solid medium and their pre-culture inliquid medium, they are seeded in the Deep Well type micro platescontaining the medium YFX50-Ac-5000 (50 g/kL of xylose and 5 g/L ofacetic acid at pH=5).

After 72 hours of fermentation in this medium, the selected strains (16in total) are those having the best growth, by analysis of the cellulardensity of the medium. Strains CNCM I-4071 and CNCM I-4829 servedrespectively as negative and positive control for the capability of thecells to use xylose for their growth.

These 16 strains were evaluated on the kinetics on this same medium. Theresults are shown in FIG. 1 which shows the mass loss as a function ofthe fermentation time. As expected, since this medium contained onlyxylose (no glucose), the observed profile for all strains tested issingle phase. On the other hand, it appears that the derivative at thebeginning of the curve varies depending on the strains. This can beformulated using the equation ax+b, where a varies according to thestrains.

FIG. 1 shows that strain E9 shows the best performance. The strain wasdeposited with Institut Pasteur (Collection Nationale de Cultures deMicroorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15) undernumber CNCM 1-4953 on Jan. 29, 2015 and was used in the remainder theexperiments.

2) Properties of Strain 1-4953:

2-1) in Xylose Medium:

The first step consisted of determining the impact of the directedevolution on the capability of this strain to resist acetic acid duringxylose fermentation. To do that, the test consisted of measuring themaximum CO₂ production rate during the xylose fermentation on a YFX50-Acmedium. In order to obtain a dose-response curve, various acetic acidconcentrations were added to the fermentation medium.

FIG. 2 shows that this strain has a maximum xylose consumption rateequal to twice the mass loss during fermentation which is about 15%greater than the rate for the strain I-4829 without acetic acid. Thisgap is even greater when the acetic acid dose is 4 g/L. In fact, at thisconcentration, the xylose consumption rate is reduced by 85% for strainI-4829, compared to only 15% for strain I-4953. This means that in thepresence of 4 g/L acetic acid and at pH 5, strain I-4953 has the samexylose consumption rate as strain I-4829 without acetic acid.

2-2) in Glucose+Xylose Medium:

In order to better understand the impact of the directed evolution onthe fermentation in general, the mass loss as a function of fermentationtime was tracked for a medium containing glucose, xylose and aceticacid.

The results are shown in FIG. 3 which clearly shows the double effect ofacetic acid on yeasts capable of fermenting xylose:

-   -   During the first phase, called the “glucose phase”, strain        1-4538 shows a fermentation initiation delay of about 24 hours.        For the other strains (I-4749, I-4829 and I-4953), the lag is        only 5 to 6 hours. This observation means that the isolated        strain has not lost resistance to acetic acid during glucose        fermentation.    -   When xylose fermentation picks up the relay, the kinetics of        strains I-4538, I-4749 and I-4829 are comparable. In contrast,        for strain I-4953, it is notable that CO₂ production (and        consequently ethanol production) is faster and greater than that        observed for the 3 other strains.

These results confirm that a strain more resistant to acetic acid duringxylose fermentation was obtained and that it is therefore globally moreeffective.

III) Conclusions:

With the method described, it was therefore possible to select at leastone stable strain, I-4953, which retained its capability for fermentingglucose with a limited initiation delay, while showing improved xylosefermentation kinetics in the presence of acetic acid.

The invention claimed is:
 1. A yeast strain characterized in that it wasdeposited at the CNCM (Collection Nationale de Cultures deMicroorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 ParisCedex 15) under number 1-4953 on Jan. 29,
 2015. 2. A yeast obtained byculturing the strain according to claim
 1. 3. The yeast strain accordingto claim 1, further comprising one or more genetic modifications whichdo not negatively impact xylose metabolism in the presence of aceticacid.
 4. A method for producing fermentation products or ethanolcomprising the following steps: Incubation of a material or mediumcontaining xylose with the yeast strain according to claim 1;Fermentation under anaerobic or semi-anaerobic conditions; and Recoveryof the one or more fermentation products, or ethanol.
 5. The methodaccording to claim 4, characterized in that the material or medium alsocontains glucose.
 6. The yeast strain of claim 1, further comprising atleast one copy of an exogenous gene encoding xylose isomerase.
 7. Theyeast strain according to claim 1, further comprising at least onesupernumerary copy of a GAL2 gene.
 8. The yeast strain of claim 1,further comprising a modification which suppresses aldose reductaseactivity.
 9. The yeast strain according to claim 1, further comprisingat least one supernumerary copy of a xylulokinase (XKS1) gene or amodification of a promoter resulting in overproduction of xylulokinase.10. The yeast strain according to claim 1, further comprising at leastone supernumerary copy of a gene in the pentose phosphate pathway or amodification of a promoter resulting in overproduction of a gene productin the pentose phosphate pathway.
 11. The yeast strain according toclaim 10, wherein the gene of the pentose phosphate pathway is RPE1,RKL1, TKL1, TAL1, or a combination of any of the foregoing.
 12. Theyeast strain according to claim 1, comprising a modification resultingin the absence of xylose reductase activity in the yeast.
 13. The yeaststrain according to claim 12, comprising a mutation in a XR or XYL1gene.
 14. The yeast strain according to claim 1, comprising amodification that enables the yeast to ferment arabinose.